Poster Presentations: P4 P893

Background: DSP-8658, a novel selective peroxisome proliferator-acti-

vated receptor (PPAR)a/gmodulator, is a Phase I clinical stage drug candi-

date for the treatment of Alzheimer’s disease (AD). We have previously

reported that DSP-8658 enhances the microglial uptake of amyloid b-pepti-

de(Ab), which is a proposed trigger of the onset of AD, and also improves

cognitive function in APP/PS1 transgenic mice. The present study describes

the acute or subacute symptomatic effects of DSP-8658 on cognitive func-

tion and the results in 104-week rat carcinogenicity study.Methods: To in-

vestigate procognitive effects of DSP-8658 at 1-30 mg/kg, p.o., we

performed a mouse object recognition test, a mouse Y-maze test and a rat

passive avoidance test. To clarify the safety profile of DSP-8658 more,

the 104-week carcinogenicity study of rats was conducted. Results: DSP-

8658 (30mg/kg) significantly prevented the object memory loss in aged

mice. DSP-8658 (3 mg/kg) also improved the spatial working memory im-

pairment caused by LPS-induced neuroinflammation in mice. In the rat pas-

sive avoidance test, DSP-8658 (10mg/kg) significantly ameliorated

scopolamine-induced memory impairment. On the other hand, Wy14643

(PPARa agonist) and Rosiglitazone (PPARg agonist) also showed procog-

nitive effects, but those maximum effects were less than that of DSP-

8658. These results indicate that DSP-8658 has a great potential to restore

the memory deficits caused by aging, cholinergic dysfunction and inflam-

mation, which are the important characteristic features of AD pathology.

The potent efficacies of DSP-8658 on procognitive function, which is supe-

rior to those of the single agonists of PPAR subtypes, may be mediated by its

dual agonistic activities on both PPARa and g. In the 104-week rat carcino-

genicity study, DSP-8658 induced tumors only at the highest doses produc-

ing AUC�10-fold therapeutic exposures with the maximum recommended

human dose, and had enough cardiovascular safety margin, indicating that

DSP-8658 exhibits good safety profiles as a PPARmodulator.Conclusions:

Based on the nonclinical pharmacological and safety data, DSP-8658 has

a highly promising therapeutic profile as a drug for both symptomatic and

disease-modifying treatment of AD. DSP-8658 is expected to be efficacious

for AD in the following clinical trials.

P4-430 ANTISENSE OLIGONUCLEOTIDE-MEDIATED

INHIBITION OF MIR-33 IN CULTURED NEURONS,

ASTROCYTES AND MICROGLIA: EFFECTS ON

ABCA1 EXPRESSION, APOE LIPIDATION,

CELLULAR CHOLESTEROL AND BETA-

AMYLOID NEUROTOXICITY

Asad Qureshi1, Joanna Karasinska1, Martin Kang1, Achint Kaur1,

Piers Ruddle1, Sonia Franciosi1, Michael Hayden1, 1University of British

Columbia, Vancouver, British Columbia, Canada. Contact e-mail: jaan.

asad@gmail.com

Background: Increasing the expression of ATP-binding cassette transporter

A1 (ABCA1) reduces plaque deposits and improves memory performance

in mouse models of Alzheimer’s disease (AD). This has predominantly

been attributed to ABCA1-mediated lipidation of apolipoprotein E (apoE)

in the brain and facilitating amyloid clearance. Despite the beneficial effects

of LXR agonists - therapeutic compounds that increase ABCA1 expression-

in preclinical studies, their utility for the treatment of AD is limited by se-

rious side effects underscoring the need for discovering alternative

therapeutic approaches. MicroRNAs (miRNAs) are endogenous small, non-

coding RNA molecules that bind to target mRNAs leading to translational

repression. MicroRNA-33 (miR-33) regulates the expression of key genes

involved in cellular cholesterol metabolism including ABCA1 and has

emerged as a promising target in experimental models of atherosclerosis

and diabetes. The objective of this study is to evaluate the effects of increas-

ing ABCA1 in brain cells by inhibiting miR-33 and study the cellular and

molecular mechanisms relevant to AD pathogenesis. Methods: Antisense

oligonucleotides (ASOs), primary neurons, astrocytes and microglia, west-

ern immunoblotting, cellular cholesterol, cholesterol efflux, phagocytosis

and cytokine release assays, neuronal viability assays, intracerebroventric-

ular infusion, APP/PS1 mice and mouse neuropathology. Results: Treat-

ment of cultured neurons, astrocytes and microglia with an ASO targeting

miR-33 increases ABCA1 expression in a concentration dependant manner.

This is accompanied by increased cholesterol and ApoE efflux by astrocytes

into the cultured medium. Interestingly, increasing ABCA1 expression did

not change the total cellular cholesterol content in neurons and astrocytes,

neither did it protect neurons against Ab toxicity. Moreover, i.c.v. infusion

of miR-33 ASO also increased hippocampal and striatal ABCA1 expression

in wild type mice. Conclusions: We demonstrate that ASO mediated inhi-

bition of miR-33 can successfully be employed towards increasing ABCA1

expression in cultured brain cells and in vivo. Our data suggest that increas-

ing ABCA1 expression enhances cholesterol and ApoE efflux, which may

indirectly promote amyloid clearance. It remains to be determined whether

miR-33 inhibition affects APP processing or has any beneficial effects on

the development and progression of AD like neuropathology in relevant an-

imal models. We are confident that these findings will have significant im-

pact on the development of novel therapies for AD.

P4-431 IMPROVED PHOSPHOTAU SRM ASSAY

SENSITIVITY ENABLES MULTISITE TAU

PHOSPHORYLATION QUANTITATION IN

A PRECLINICAL MODEL OFALZHEIMER’S

DISEASE TREATEDWITH NOVEL SMALL-

MOLECULE INHIBITORS OF CASEIN KINASE 1

DELTA

Emma Lahert,Claire Russell, Ian Pike, MalcolmWard, Proteome Sciences

Plc, London, United Kingdom. Contact e-mail: claire.russell@proteomics.

com

Background:Neurofibrillary tangles, comprised of paired helical filaments

(PHFs) of hyper-phosphorylated tau, are a pathological characteristic of

Alzheimer’s disease (AD). We previously characterised PHF tau isolated

from post-mortem AD brain tissue by mass spectrometry. Subsequently,

we developed the Phospho-Tau SRM assays to measure tau phosphorylation

levels in preclinical AD models. Methods: The Phospho-Tau SRM 6plex

assay enables the quantitation of total tau plus five phosphorylation sites;

distinct human and mouse pThr181 measurements, pSer199, pThr231,

pSer262 and pSer396 (human 2N4R numbering). The Phospho-Tau SRM

7plex assay quantifies six phosphorylation sites, pSer46, pThr50,

pSer113, pSer396, pSer404, pSer433, as well as distinct measurements cov-

ering the R406W mutation, present in the TMHT tau transgenic mouse

model. Prior to SRM analysis, phosphopeptides were resolved by microflow

(100mL/min) reversed phase chromatography (XBridge C18 3.5mM, 1.0 x

100mm, Waters). The linear working range of the microflow-PhosphoTau

SRM 6plex assay was 5-1000fmol on column (o/c), with CVs ranging

from 5-20%. Per analysis up to 10mg total protein per samplewas consumed.

Results:Herein we describe theminiaturisation of the PhosphoTau SRM as-

says to nanoflow (200nl/min; Easy C18 3mM ID 75mM x 100mm, Thermo-

Fisher). Preliminary data demonstrates a 50 fold improvement in linear

working range, down to 100 atmol o/c, and detection of endogenous Tau

phosphorylation levels in preclinical ADmodels from as little as 1mg of ma-

terial. Conclusions: The utility of the nanflow-PhosphoTau SRM assays to

quantify multiple site specific phosphorylation events will be demonstrated

in a tau transgenic mouse model treated with novel small molecule inhibi-

tors of Casein Kinase 1 delta.

P4-432 SMALL MOLECULES TARGETING RHO GTPASE

SIGNALINGALTERED INALZHEIMER’S DISEASE

Qun Lu1, Amy Friesland1, Jonathan Lee1, Yan-Hua Chen1, 1The Brody

School of Medicine at ECU, Greenville, North Carolina, United States.

Contact e-mail: luq@ecu.edu

Background: Altered intracellular protein transport and synaptic remodel-

ing are two of the most prominent cellular processes that are affected in Alz-

heimer’s disease (AD) as well as other neurodegenerative diseases. Rho

subfamily of the small GTPases within the Ras superfamily is essential

for modulating theses functions. Research has increasingly established the

RhoA, Rac1, and Cdc42 subclasses of small G-proteins as controlling ele-

ments of secretase trafficking, Ab production, neurotransmitter receptor sig-

naling, and the synaptic cytoskeletal dynamics. Understanding how they

operate in the context of memory and learning is pivotal for the development

Poster Presentations: P4P894

of Rho-GTPase based therapeutic treatments of AD. Methods: Our recent

studies demonstrated the remarkable alteration of actin cytoskeleton and

its regulatory proteins in AD. These proteins include filamin (Lu et al.,

JAD, 2010), G-actin, and components of Rho-GTPase signaling. In order

to establish a repertoire of small molecule modulators of Rho-GTPase sig-

naling, we performed a computer-assisted in silico screening of the SPECS

database using GLIDE program (Schrodinger) for chemical compounds that

can disrupt the interaction of Cdc42 with intersectin (Friesland et al., PNAS,

2013). This study led to the discovery of a number of small molecules tar-

geting Rho GTPases including Cdc42, RhoA, and Rac1. Results: The bio-

chemical and imaging studies not only established the first small molecule

ZCL278 targeting Cdc42-intersectin interaction, it also provided the proof-

of-concept results showing the effectiveness of ZCL278 to interfere with

Golgi organization and neuronal cytoskeleton. Additional studies on ZCL

series compounds showed favorable drug tolerance, potential CNS effects,

and distinct neuronal responses to staurosporine and glutamate induced neu-

ronal toxicity. Our other studies employ isolated primary cortical neurons

from triple transgenic (3xTg) mice expressing AD-like mutations in presen-

lin 1 (M146V), APP (695swe),and Tau P301L. In the3xTg neurons, filamin,

G-actin, and Cdc42 showed redistribution in the soma and the axonal orden-

dritic processes, further validating the potential significant involvement of

actin cytoskeleton in AD pathogenesis. Conclusions: The discovery of

ZCL series chemicals could form a novel compound base to potentially de-

velop into AD modifying therapeutics.

P4-433 THE EFFECT OFACUTE ADMINISTRATION OF

LEVETIRACETAM ON CEREBRAL PERFUSION IN

ALZHEIMER’S DISEASE AS MEASURED BY

ARTERIAL SPIN LABELING MRI

Daniel Press1, Weiying Dai1, Jocelyn Breton1, Susan Herman1,

David Alsop1, 1Beth Israel Deaconess Medical Center, Boston,

Massachusetts, United States. Contact e-mail: dpress@bidmc.harvard.edu

Background: Both animal models of Alzheimer’s disease (AD) and a study

in mild cognitive impairment suggest that neuronal hyperexcitability, possi-

bly epileptic in nature, occur in AD. We have previously shown that medial

temporal lobe perfusion as measured by arterial spin labeling (ASL) MRI is

increased in mild AD. To test whether this increase in perfusion might be

due to neuronal hyperexcitability, we used the acute administration of lev-

etiracetam (LEV), a medication that selectively blocks epileptic discharges,

as a pharmacological probe. Methods: Patients with mild AD (n¼10 en-

rolled, n¼7 completed, 3 did not complete the protocol due to claustropho-

bia and excessive motion) underwent ASL MRI in three sessions held one

week apart, performed in a double-blindmanner and counterbalanced for or-

der. At each session, a 32-lead EEG was recorded before, during, and after

i.v. administration of placebo, levetiracetam 2.5mg/kg or levetiracetam

7.5mg/kg. Regional CBF was then measured with ASL MRI and cognitive

performance on a battery of tests was performed. CBFwas normalized to the

whole brain mean and changes were expressed as percent change from pla-

cebo-baseline with analysis via paired t-tests. Results: The acute adminis-

tration of levetiracetam lead to significant decreases in perfusion

bilaterally in the precuneus (mean +/- SEM: -5.3% +/- 1.5% in the left

and -5.6% +/- 1.3% in the right, p< 0.05, uncorrected) and increases in per-

fusion in the amygdala/anterior temporal lobes (9.9% +/- 3.0% in the left

and 10.3 +/- 3.4% in the right, p < 0.01, uncorrected). Cognitive testing

showed little change at the low dose of LEVand a trend towards worse per-

formance at the higher dose. Conclusions: The acute administration of

i.v. levetiracetam lead to a pattern of relative decreased perfusion in poste-

rior parietal regions and relative increased perfusion in anterior temporal

lobe regions. The presence of significant changes in CBF after drug

administration suggests that LEV is modifying neuronal activity. The rela-

tive CBF increase in anterior temporal lobe and decreases in posterior cin-

gulate/precuneus regions could represent differential effects directly on

hyperexcitability vs. indirect changes related to reduced inhibitory network

activity.